Original Citation:
Development and validation of an HPLC-MS/MS method for the detection of ketamine in Calliphora vomitoria (L.) (Diptera: Calliphoridae)
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DOI:10.1016/j.jflm.2018.04.013 Terms of use:
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Development and validation of an HPLC-MS/MS method for the detection of ketamine in Calliphora vomitoria (L.)
(Diptera: Calliphoridae)
Journal: Journal of Medical Entomology Manuscript ID JME-2017-0328
Manuscript Type: Research Article Date Submitted by the Author: 12-Oct-2017
Complete List of Authors: Magni, Paola; Murdoch University School of Veterinary and Life Sciences, Department of Molecular and Biomedical Sciences
Pazzi, Marco; Università di Torino, Dipartimento di Chimica Droghi, Jessica; Università di Torino, Dipartimento di Chimica Vincenti, Marco; Università di Torino, Dipartimento di Chimica
Dadour, Ian; Boston University School of Medicine, Program in Forensic Anthropology, Department of Anatomy & Neurobiology
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from the list</b>: Direct Injury, Myiasis, Forensics Field Keywords: Forensic Entomology, Toxicology Organism Keywords: Calliphoridae
Abstract 1
2
Entomotoxicology studies the detection of drugs or other toxic substances from insects developing
3
on the decomposing tissues. Entomotoxicology also investigates the effects of these substances
4
on insect development, survival and morphology to provide an estimation of the minimum time
5
since death. Ketamine is a medication mainly used for starting and maintaining anesthesia.
6
Ketamine is also used as a recreational drug and as a sedating drug to facilitate sexual assault,
7
resulting in several deaths. Furthermore, ketamine has been also implicated in suspicious deaths
8
of animals. The present study describes for the first time the development and validation of an
9
analytical method suited to detect ketamine in Calliphora vomitoria L. (Diptera: Calliphoridae),
10
using liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). This study also
11
considers the effects of ketamine on the survival, developmental rate and morphology of C.
12
vomitoria immatures. Larvae were reared on substrates homogeneously spiked with ketamine
13
concentrations consistent with those found in humans after recreational use (300 ng/mg) or
14
allegedly indicated as capable of causing death in either humans or animals (600 ng/mg). The
15
results demonstrated that (a) HPLC-MS/MS method is applicable to ketamine detection in C.
16
vomitoria immatures, not adults; (b) the presence of ketamine at either concentration in the food
17
substrate significantly delays the developmental time to pupal and adult instar; (d) the survival of
18
C. vomitoria is negatively affected by the presence of ketamine in the substrate; (e) the length and
19
width of larvae and pupae exposed to either ketamine concentration were significantly larger than
20
the control samples.
21 22
Keywords: Entomotoxicology, ketamine, HPLC-MS/MS, blowflies 23 24 25 26 27 28
1. Introduction 29
30
In the process of an investigation regarding a suspicious death, toxicological analysis sometimes
31
plays a pivotal role in identifying the presence of chemical substances that may have caused death
32
directly (e.g. overdose) or indirectly (e.g. altering the state of awareness) (1). Historically, blood
33
and urine represent the most commonly used biological matrices for the identification of the
34
alleged intoxicating substances (simply referred to as “drugs” in this study) in both the living and
35
the dead. However, over the last few years forensic toxicologists have focused their attention on
36
the use of non-conventional biological matrices, with the aim of making the sampling less invasive
37
and more readily available, (2). The criteria used for the selection of non-conventional matrices
38
must be correlated with the aim of the investigation, the ease of sampling, the cost of the analyses,
39
the reliability and reproducibility of the results, and the overall analytical complexity (2). Among the
40
non-conventional biological matrices, the majority of studies have focused on keratine (hairs and
41
nails), sweat, saliva, amniotic fluid and meconium (2).
42
The insects found on a highly decayed or skeletonized corpse can also be included as a
non-43
conventional matrix, useful in the identification of drugs, metals, pesticides and poisons. The
44
discipline of entomotoxicology involves the combination of entomology and toxicology by
45
considering both the presence of the toxic substances in the insects that colonized the remains
46
and their effects on the insects’ survival and development rate (3). In a forensic context, especially
47
when the toxicological analyses have to be conducted on highly decomposed tissues, it has been
48
demonstrated that the use of necrophagous insects provides higher sensitivity and better results
49
compared to decomposed tissues (4, 5). Furthermore, by studying the effects of the drugs on the
50
insects it is possible to apply appropriate correction factors to pre-existing tables of growth
51
concerning the insects’ morphology or survival rate, and obtain a more focused estimation of the
52
minimum time since death (minPMI) (6). Overall, entomotoxicological studies may provide
53
information regarding both the cause and the time of death.
54
Ketamine, 2-(2-chlorophenyl)-2-(methylamino)-cyclohexanone, is an arylcycloalkylamine
55
structurally related to phencyclidine (PCP) and cyclohexamine. It was synthesized in 1962,
patented in 1963, and tested on human prisoners in 1964, with the outcome of it being a more
57
favourable choice over PCP as a dissociative anaesthetic. After its approval in 1970, it was
58
administered as an anaesthetic to American soldiers during the Vietnam War (7-9). At present,
59
ketamine is a medication with unique therapeutic value in veterinary medicine, mainly used for
60
inducing and maintaining anaesthesia (known under the name of Ketalar, Ketaminol Vet.,
61
Clorketam, Imalgene, Anesketin, Ketamine Ceva, Vetalar Vet., Narketan, Ketaset), and, to a lesser
62
extent, it is used in human medicine especially in paediatric surgery (Ketalar, Ketamine
63
Panpharma, Ketolar, Ketanest-S) (10). Nonmedical use of ketamine began in the 1970s, but it was
64
not until 1999 that ketamine was introduced into the U.S. Food and Drug Administration register
65
(11). Known also with the street name Special K, K, ket, kitkat, super k, horse trank, tac et tic, cat
66
Valium, and vitamin K, ketamine is illegally used for its hallucinogenic effects, that cause the user
67
to see, hear, smell, feel, and taste non-existing entities different from reality (12). Ketamine also
68
shows dissociative effects, causing a feeling of disconnection between the mind and the body in
69
the user (‘out-of-body experience’) (10). The literature reports a number of accidental/sudden
70
death cases in which ketamine was used, alone or in combination with other drugs, e.g. cocaine,
71
amphetamine, cannabis, or alcohol (10, 13). Ketamine has also been used in a number of
drug-72
facilitated sexual assaults and was implicated in several deaths globally (14). To note, ketamine
73
has also been associated with the suspicious deaths of animals (e.g. sedation with a wrong dose
74
of the drug) and in cases of animal cruelty (15).
75
Within the entomotoxicological literature (16), only two studies have addressed the effects of
76
ketamine on blowflies (17, 18). Lü et al. (17) investigated the effects of ketamine on Chrysomya
77
megacephala’s (Fabricius) (Diptera: Calliphoridae) larval lengths, weights, and the developmental
78
duration of larval instar, but no analytical method was developed to identify the presence of
79
ketamine in the insects. Zou et al. (18) detected the presence of ketamine in Lucilia sericata
80
(Meigen) (Diptera: Calliphoridae) by gas chromatography-mass spectrometry (GC-MS), and they
81
also observed the effects of ketamine on the development and morphology of this fly. However, the
82
analytical method proposed by Zou et al. (18) did not take into account all the standard parameters
83
suggested by the international scientific standards for validation.
The present study describes the development and validation of an analytical method based on
85
liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), suitable to detect ketamine in
86
immatures of Calliphora vomitoria L. (Diptera: Calliphoridae). Furthermore, the effects of the
87
presence of ketamine were examined on the developmental time, the morphology (length and
88
width) and survival of C. vomitoria immatures, reared on a substrate spiked with the drug.
89 90
2. Material and Methods 91
92
2.1. Preparation of foodstuff and rearing of C. vomitoria 93
Calliphorids (Diptera: Calliphoridae) are blowflies widely distributed in the different continents.
94
Many species known to be early coloniser of dead bodies, and therefore they are used in forensic
95
entomology for the estimation of the minPMI (20). C. vomitoria is distributed throughout the
96
Holarctic region and it is mainly present in rural areas during the cold season (21-23). This fly was
97
chosen for this study, as it is one of the most common species found in cases of forensic relevance
98
(24).
99
Colonies of C. vomitoria were reared following the procedures described by Magni et al. (6, 25),
100
starting from wild flies caught in several rural areas of the north west of Italy. Wild flies were
101
identified by a taxonomist and regularly added to colonies to prevent inbreeding (21). As in
102
previous research, C. vomitoria used in this experiment were harvested from a third generation
103
laboratory culture. Adults were provided with distilled water and sugar ad libitum for their
104
sustenance (from eclosion to the end of the experiment), while beef liver was provided as a
105
medium for the development of oocytes (introduced on day 5 after eclosion and left 48 hours in the
106
cage) and to obtain eggs (introduced on day 12 after eclosion) (25). The liver was checked every 2
107
hours and following oviposition, 3 egg clusters containing approximately 1000 eggs (1.2 g) were
108
deposited with a fine paintbrush onto beef liver aliquots (500 g x 3) already spiked with ketamine at
109
variable concentration levels and homogenised (control 0 ng/mg, 300 ng/mg, 600 ng/mg – simply
110
referred as C, T1, T2 respectively). The amounts of ketamine chosen to spike the substrate were
111
based on the concentrations found in humans after recreational use (300 ng/mg) or that which has
been indicated as capable of causing death in either adult humans (≈80 Kg) or animals of the
113
same size (600 ng/mg) (13, 19). Liver was used as the fly food substrate because (a) it is the
114
typical medium for forensic entomology experiments (26, 27); (b) it was used in previous research
115
on ketamine and blowflies (18); (c) it is one of the tissues in which ketamine distributes first and its
116
metabolic evolution starts (28). Experimental livers were homogenized with increasing volumes (0,
117
12.5, 25, 37.5, 50, 75, and 100 µL) of methanol solution of ketamine (1 mg/L) to reach the final
118
concentration. Homogenization was performed using a A11 basic Analytical mill (IKA®-Werke
119
GmbH & Co.). Following laboratory standards, a T18 digital ULTRA-TURRAX (IKA®-Werke GmbH
120
& Co.) was used, to obtain a uniform distribution of the analytical standard. Each experimental liver
121
was placed on a round plastic tray (Ø 14 cm with moistened paper on the base to prevent
122
desiccation) with high sides (10 cm) to observe the start of the larvae post-feeding instar. Each
123
plastic tray was placed on top of 5 cm of dry sand within a larger plastic box (22x40x20 cm) which
124
was covered with a fine mesh cloth and sealed using an elastic band. Sand was used to facilitate
125
pupation. Immature and adult flies were reared at 23.3 ± 1.2°C laboratory temperature with
126
approximately 20% RH and a photoperiod (h) of 12:12 (L:D). Temperature data in this study were
127
recorded using Tinytag® data-loggers with data being recorded every hour.
128 129
2.2. Sample collection 130
Two samples, one consisting of 30 individuals and another amounting to 1 g from each treatment
131
were collected when C. vomitoria reached the second (L2), third (L3), post-feeding (PF) pupal (P)
132
and adult (A) instars. Empty puparia (EP) were also collected.
133
Each sample of 30 individuals was used for morphological analyses. Specimens were sacrificed by
134
immersion in hot water (>80°C) for 30 seconds and preserved in 70% ethanol (29). Following
135
preservation, larvae and pupae were measured with a digital calliper (Terminator®) under a
136
stereomicroscope (Optika SZM-2). As described by Day and Wallman (30) the length of each larva
137
was measured between the most distal parts of the head and the eighth abdominal segment, while
138
the width of each larva was measured between the ventral and dorsal surfaces at the junction of
the fifth and sixth abdominal segments. Regarding the pupa, the length was measured between
140
the most distal parts, while the width was measured in the largest part of the pupal case.
141
Each sample weighing 1 g from each of the instars was stored at -20°C until the sampling period
142
finished and then they were analysed to detect ketamine. Larvae of L2 and L3 instars were
143
sacrificed and stored only after careful cleaning of each individual with water and neutral soap to
144
remove any external contamination. Adults were not provided with any food or water source and
145
were sacrificed 2 days after their emergence. The analytical method was validated using 50 mg of
146
control EP, chosen as the target matrix because of their high chitin content. Empty puparia were
147
also chosen because they can be found at the scene for a much longer period after emergence,
148
and in such circumstances they may represent the only reliable sample for toxicological analyses
149
(31).
150
When the larvae reached the PF instar, 100 individuals from each treatment were placed in
151
separate boxes. The time to pupation, the total number of pupated individuals, as well as the time
152
to eclosion and the total number of emerging adults were recorded.
153 154
2.3 Toxicological analysis 155
156
Chemicals and reagents – Liquid ketamine (≥99%) and d4-ketamine 100 µg/mL in methanol (as 157
free base) ampule of 1 mL, certified reference material Cerilliant® were purchased from Sigma
158
Aldrich® (Milano, Italy). Standard solutions of ketamine in CH3OH (0.5 mg/L, 1 mg/L, 10 mg/L, 100 159
mg/L, 1000 mg/L) and d4-ketamine (used as the internal standard, ISTD) in CH3OH (10 mg/L and 160
1 mg/L) were prepared from the pure liquid standards. Dichloromethane (CH2Cl2), methanol, 161
trifluoroacetic acid were also purchased from Sigma Aldrich® (Milano, Italy).
162 163
Sample preparation HPLC-MS/MS analysis – Larvae (L2, L3, PF), P, EP and A samples were
164
placed separately in falcon tubes (50 mL) and dichloromethane was added as part of the
165
preliminary wash. The tubes with larvae and pupae were then placed in a vortex for two minutes
166
and the organic solvent was discarded. Meanwhile, the EP were dried at room temperature under
nitrogen. Following crystallisation using liquid N2, they were crushed with a glass rod and a 50-mg 168
aliquot was placed in a new tube. To validate the method, control C. vomitoria EP were spiked with
169
different amounts of ketamine at this stage, by adding different volumes (0, 12.5 , 25 , 37.5 , 50,
170
75 , and 100 µL) of methanol solution of ketamine (1 mg/L). In addition, 2 ml of CH3OH was added 171
and 10 mL of d4-ketamine (10 mg/L in CH3OH) solution was added as the ISTD. The tubes were 172
sealed and placed in heating-blocks at 60°C to extract/dissolve the matrix, for 4 hours. After
173
elimination of the solid residues, at the digest sample was added trifluoroacetic acid (30µL) then
174
the sample was dried at 70°C under nitrogen stream. After drying, the analytes were recovered
175
with 200 µL of methanol. 10 µL of the solution was injected into the HPLC-MS/MS instrument.
176 177
HPLC-MS/MS analysis – Analytical determinations for the detection of ketamine was performed
178
with LC Varian 920 coupled with Varian 320 MS operating in the electrospray ionization mode.
179
Samples (10 µL ) were injected into a Luna C18, 150mm x 2mm x 3 µm, with C18 precoloumn filter
180
(Security Guard, Phenomenex Inc., Torrance, CA-US). Eluition mixture was composed by 87%
181
formic acid 0.1% and 13% acetonitrile 0.1%. Temperature of drying gas was 200°C and
182
nebulization temperature was 55°C, electron multiplier potential was 1500V. In order to complete
183
the quantitative analysis, the mass analyzer was operated in Multiple Reaction Monitoring (MRM)
184
and transition followed to identify ketamine were reported in Table 1.
185 186
Method validation – Ketamine detection method was validated according to the guidelines of
187
Raposo (32), the ISO/IEC 17025 requirements and ICH guidelines (33, 34). The validation protocol
188
included quantitative determination of ketamine in larvae, P, EP and adults: specificity, linearity,
189
back calculation, limit of detection (LOD), limit of quantitation (LOQ), extraction recovery (ER%),
190
repeatability, matrix effect and carry over were determined.
191 192
Specificity – Ten samples of the control EP were used to ascertain the method’s specificity. Five
193
of them were spiked with 1 mg/L of ISTD. The specificity test was successful if the S/N ratio was
194
lower than 3 at the expected retention time of the target analytes, for all ion chromatograms.
196
Linearity – The linearity of the calibration model was checked by analyzing control EP samples
197
(50 mg x 5 repetitions for each calibration point) spiked with ketamine solution at concentrations of
198
0, 0.5, 0.75, 1, 1.5 and 2 ng/mg. d4-ketamine with a final concentration of 10 ng/mg was used as
199
the ISTD. The linear calibration parameters were calculated by least-squares regression, and the
200
correlation coefficient (R2) was used for a rough estimation of the linearity. For determination of
201
linearity were considered Mandel and Olivieri’s principles (35, 36). Another parameter used to
202
evaluate linearity was back calculation, which, from calibration curve point, calculates backwards
203
the concentration of ketamine in semple starting from the instrumental signal. Back calculation is
204
useful to evaluate calibration curve goodness. Quantitative results from area counts were
205
corrected using the ISTD signal.
206 207
Limit of detection and limit of quantitation (LOD and LOQ) – LOD and LOQ were calculated
208
according to Hubaux and Vos (37). This method is based on calibration curve so the result is more
209
relevant and sturdy to the method that has been developed than standard calculation of LOD and
210
LOQ.
211 212
Extraction recovery (ER%) – ER% was evaluated at two concentrations of ketamine in control EP:
213
0.75 and 2 ng/mg. For each of these concentrations, five samples were spiked before the digestion
214
step of the matrix and five after the extraction. ER% was calculated by the average ratio of the
215
analyte concentration determined after its extraction (first set) to the one determined on the spiked
216
extract (second set).
217 218
Repeatability (intra-assay precision) – Repeatability was calculated as the percent coefficient of
219
variance (CV%) after spiking ten samples of control EP with two concentrations of ketamine: 0.75
220
and 2 ng/mg. Repeatability was considered acceptable when CV% <20%.
221 222
Carry Over – Carry-over effect was evaluated by injecting an alternate sequence of ten blank EP
223
semples spiked with ketamine at concentration of 0.5 ng/mg and ten blank EP samples spiked with
224
ketamine at a concentration of 2 ng/mg. To ensure the absence of any carry-over effect, for each
225
transition, the signal-to-noise ratio (S/N) from negative samples had to be lower than 3.
226 227
Matrix-effect – Matrix effect was evaluated following the Matuszewski’s criteria (38) analysing five
228
EP samples (chitinic matrix) spiked with ketamine at concentration of 0.25 ng/mg and five samples
229
at 2 ng/mg both five sample of cheratin matrix at the same concentration.
230 231
2.4 Statistical analysis 232
233
Ketamine concentration in larvae, pupae and adults as well as their respective lengths and widths
234
in different treatments were analysed by one-way ANOVA and Tukey test. Pupation and eclosion
235
rate were analysed by a one-way ANOVA and Pearson’s Chi-squared test. The level of
236
significance was set at P < 0.05. Calculations were performed using IBM SPSS Statistics 22
237
statistical software package.
238 239
3. Results 240
Entomotoxicological analyses by HPLC-MS/MS confirmed the possibility that ketamine can be
241
detected in different instars of C. vomitoria reared on food substrates containing ketamine in
242 concentrations of 300 ng/mg and 600 ng/mg. 243 3.1 Method validation 244 245
The following parameters were obtained: coefficient of linearity (R2), limit of detection (LOD), limit
246
of quantitation (LOQ), extraction recovery (%), and repeatability (CV%) (Table 2). Specificity was
247
satisfactory, while no matrix effects and carry over effects were observed.
248 249
3.2 Ketamine concentration 250
251
A summary of the ketamine concentration found in the different treatments and instars of C.
252
vomitoria detected by HPLC-MS/MS is reported in Table 3.
253
HPLC-MS/MS analyses confirmed that the ketamine artificially added to the food substrate was
254
present in the different immature instars of C. vomitoria as well as in the EP. The ketamine
255
concentration was not found to be present in C. vomitoria adults analysed by HPLC-MS/MS.
256
The ketamine concentration was absent (lower than the LOD) in all the control samples, in the L2
257
of both the treatments and in all the A samples analysed by HPLC-MS/MS.
258
The peak of ketamine concentration was found in the L3 of both treatments and analytical methods.
259
Overall, ketamine shows an increase in concentration until the larvae reach L3, then a decrease in
260
the following larval instars and an increase in the P and EP. The amount of ketamine found in all
261
treatments and instars was found to be significantly different from the controls. Statistical relevant
262
differences were also found between T1 and T2 treatments (Table 3).
263 264
3.3 Growth rates and survival 265
266
The presence of ketamine in the food substrate had significant effects on fly development time,
267
especially in the time from oviposition to eclosion (Table 3). The time from oviposition to pupation
268
was similar for control larvae and for T1 larvae, but it was significantly different between control
269
larvae and T2 larvae, that needed approximately one day more to complete pupation. The time
270
from oviposition to eclosion was significantly different between control and larvae feeding on liver
271
containing the two concentrations of ketamine (1-2 days more to complete metamorphosis). The
272
difference between the treatments was not significant for either the time from oviposition to
273
pupation and oviposition to eclosion (Table 4).
274
Ketamine present in the food substrate significantly affected C. vomitoria survival during the early
275
instars of development (until the P instar), but it was only during metamorphosis that the effects of
276
the presence of ketamine were extreme. Table 4 shows that during the PF instar only a maximum
of 15% of larvae died prior to pupation (2% in C; 10% in T1; 15% in T2), while during
278
metamorphosis survival was 85% in C, 37% in T1 and 9% in T2. The survival of pupae was
279
significantly different only between the control and both the treatments, while the survival of the
280
adults was significantly different between all treatments.
281 282
3.4 Larval and pupal length 283
284
Significant differences were observed in the average length of larvae and pupae between the
285
control and treatment groups (Table 5). However, significant differences occurred only in the length
286
of advanced L3 for T2 treatment with respect to the control and T1, and in the length of P for T2
287
treatment with respect to the control. The length of L2, early L3 and PF of all the treatment groups
288
were not significantly different from control (Table 5).
289 290
3.4 Larval and pupal width 291
292
Significant differences were observed in the average width of larvae and pupae between control
293
and treatment groups (Table 6). The width of control larvae and pupae was significantly smaller
294
than T2 individuals during the whole cycle of life. Larvae of T1 were found to have a larger width
295
with respect to the control only in the advanced L3 stage, while during the PF instars were
296
significantly smaller in width with respect to the T3 individuals.
297 298
4. Discussion 299
The use of ketamine in a medical and veterinary setting has been shown to be efficient and safe.
300
However, in the recent past the abuse of ketamine has caused severe harm to individuals (39). A
301
2006 US report shows that approximately 2.3 million teens and adults have used ketamine in their
302
lifetime (40). Ketamine it is extremely popular amongst drug users at parties all over the world and
303
in the last 10 years the number of ketamine-related deaths have significantly increased (41). There
304
have been major concerns in regards to driving under the influence of ketamine and the use of this
drug to facilitate sexual assault (39).
306
The entomotoxicology literature reports only two studies which focused on the presence of
307
ketamine in the food substrate and its effects on blowfly development (17, 18). One study (17)
308
considers colonies of Ch. megacephala, a blowfly occurring in Australasia, South Africa, Southern
309
United States and South America (20), reared on food substrates spiked with different
310
concentrations of ketamine. The aim of this research was to determine the effects of ketamine on
311
blowfly development when reared at different temperatures (17). The other study (18) considers
312
colonies of the cosmopolitan necrophagous blowfly L. sericata, reared on the tissue of rabbits
313
killed following different doses of ketamine. The aims of this study were the detection of ketamine
314
in larvae by GC-MS and the observation of the effects of ketamine on the larval morphology and
315
development of L. sericata (18). The current research is the first comprehensive study regarding
316
the effects of ketamine on C. vomitoria flies reared on liver homogenised with two concentrations
317
of ketamine. The validated HPLC-MS/MS analytical procedure detected the presence of ketamine
318
in C. vomitoria larvae, pupae and empty puparia. Furthermore, ketamine artificially added to the fly
319
food substrate produces a significant increase in larval and pupal size (length and width), a
320
significant increase in the time required to complete development and a significant decrease in the
321
survival of this fly species especially during the period of metamorphosis.
322 323
Ketamine concentration – As stated, at present only two studies pertain to the effects of 324
ketamine on blowflies. However, comparisons and analogies regarding the concentration of the
325
drug in the flies can be made only with the research of Zou (18), since the other published
326
research (17, 42) lacks any toxicological analyses of the flies reared on the food substrate spiked
327
with ketamine.
328
In the research of Zou et al. (18) ketamine was identified by GC-MS in L. sericata immatures
329
(larvae only) when reared on rabbits killed after receiving an intravenous injection of ketamine at
330
different concentrations (1/4LD50, 1/2LD50, LD50, 2LD50). Rabbit liver and muscle containing
331
different amounts of ketamine were used as food to rear the fly colonies. Results show that
332
ketamine concentrations were more consistent (higher in concentration and present in several
immature instars) in treatments that had liver as food rather than muscle tissue. This is the most
334
parsimonious explanation because (a) following GC-MS analyses of both the organs, ketamine
335
was found to have a higher concentration in liver than in muscles and (b) liver is the organ in which
336
ketamine metabolism occurs (28). To note, the analytical method used in the research of Zou et al.
337
(18) was validated only for linearity and only 10 larvae at the different instars were used for the
338
toxicological analysis. As well this sample consisted of 10 larvae aging from 12 to 120 hours (from
339
L2 instar to P instar) which is not consistent in terms of analytical weight and drug content. In order
340
to obtain reliable results, the same amount of sample should be used at the same life stage
341
throughout the experiment.
342
In the present research, ketamine was identified by HPLC-MS/MS and the analytical method was
343
validated following a set of international standards (33, 34). During the study 1 g of insect material
344
at each instar was used as a sample for the toxicological analyses. Ketamine was detected by
345
HPLC-MS/MS in all immature instars and pupal remains of C. vomitoria, Negative results in C.
346
vomitoria adults were surprising, because it is known that upon emergence as an adult, the flies
347
rapidly eliminate the drug introduced with the diet during the immature life stages (42, 43). Lastly,
348
accordingly, to Zou et al. (18) ketamine was present in higher concentrations in larvae of the
349
treatments with higher concentration of ketamine and no metabolites of ketamine were detected.
350 351
Effects of ketamine on fly growth rate and survival – C. vomitoria growth rate is affected by the 352
presence of ketamine in the food substrate. In the treatment with recreational-use concentration
353
(T1) only the time of metamorphosis was affected by the presence of the drug, while in the higher
354
dose treatment both the period needed to reach the pupation and metamorphosis were affected.
355
These results are in agreement with the findings regarding the effects of different ketamine
356
concentrations on Ch. megacephala (17). However, they are in contrast with findings regarding L.
357
sericata reared on different ketamine concentrations that showed a delay in the early development,
358
but an overall reduction of the time needed to reach the pupal stage (18). Furthermore,
359
Parasarcophaga ruficornis (Fabricius) (Diptera: Sarcophagidae) reared on different concentrations
360
of PCP, another dissociative drug similar to ketamine, showed no significant difference in the larval
growth when comparing control vs treatment groups (42).
362
When considering survival data the only available information regarding ketamine and blowflies
363
demonstrates that by increasing the ketamine dosage in the food substrate the survival of C.
364
vomitoria will decrease, especially during the period of metamorphosis. A similar trend was
365
observed in P. ruficornis reared on different concentrations of PCP (42).
366
All previous research (17, 18, 42) underlines how similar drugs can play a role in the physiology of
367
different fly species, but before such assertions, the limitations of these studies regarding the lack
368
of repetition needs to be addressed
369 370
Effects of ketamine on larval and pupal length and width – Lü et al. (17) analysed the length of 371
Ch. megacephala reared on food substrates containing ketamine in doses associated with
372
1/2LD50, LD50, 2LD50 for an adult male of approximately 70 kg. It is important to note that larval
373
samples were sacrificed with a 50:50 v/v blend of ethanol and xylene and preserved in 75%
374
alcohol (17). This preservative method makes the estimation of real length difficult to compare due
375
to larval shrinkage. It is not the method recommended as a standard of best practice in forensic
376
entomology (44). Regardless of the preservation method used by Lü et al. (17), this research
377
showed that the relative average length of Ch. megacephala larvae in all the treatment colonies
378
was significant less than the control for larvae between 16 to 64 hours (= until the L3). However,
379
since the overall duration of the PF instar of the treatments was longer compared with the control,
380
the PF Ch. megacephala larvae in all the treatment colonies were significantly larger in length
381
compared to the control. These results, however, are not absolute measures and cannot be
382
compared with this study (47).
383
In the present research as well as in the research of Zou et al. (18) fly immatures are preserved
384
according to the standards and guidelines for forensic entomology, by sacrificing specimens in hot
385
water and preserving them in 70% ethanol (44). Similarly, the two studies show that larvae reared
386
on substrates enriched with ketamine are significantly longer in length when compared to the
387
respective controls (18).
388
In the present research the width of larvae and pupae was also considered. The length of fly larvae
is often used to help provide an entomological estimate of the minPMI, but the curved shape of the
390
larvae can affect the accuracy of length measurements. The width is not affected by the curved
391
shape of the larvae, it has been demonstrated to be comparable with body length for larval age
392
prediction (30) and it has been used in previous entomotoxicology research (48). Despite the width
393
measurement not often being used to measure larvae size, this data was considered in the present
394
research as it provided a comparison with the control treatment. Statistical results on C. vomitoria
395
showed that larvae and pupae reared on substrates enriched with ketamine are significantly larger
396
in width compared to the control. As a consequence, when ketamine is present in the food
397
substrate both width and length can be taken to estimate the age of immatures with larval width
398
being a more accurate.
399 400
5. Conclusions 401
Although ketamine is important in medical and veterinary practice, it is also illegally used by
402
humans to hallucinate and to facilitate sexual assaults. It has been a drug of choice found amongst
403
some high profile investigations, e.g. the death of the world famous singer Amy Winehouse (2011).
404
This research validates an analytical method based on HPLC-MS/MS to detect the presence of
405
human recreational and lethal doses of ketamine in blowflies.
406
This research shows that C. vomitoria immature and adults accumulate ketamine and that the
407
development and survival of C. vomitoria feeding on liver containing ketamine can be significantly
408
affected by the presence of the drug.
409
This research underlines the need of further entomotoxicology studies, such as: a) effects of
410
ketamine on different fly species reared at different temperature; b) the effects of “ketamine
411
cocktails” on blowflies; c) the effects of ketamine on subsequent generations; d) the validation of
412
alternative analytical methods (e.g. GC-MS) with the aim of allowing laboratories in possession of
413
other analytical techniques to benefit from this type of research.
414 415 416 417
References 418
419
1. Bowdle TA, Radant AD, Cowley DS, Kharasch ED, Strassman RJ, Roy-Byrne PP.
420
Psychedelic effects of ketamine in healthy volunteers. Anesthesiol 1998. 1998;88:82-8.
421
2. De Giovanni N, Fucci N. Le matrici biologiche non convenzionali in tossicologia forense.
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Diagnostica medico-legale. Roma: ARACNE Editrice A.r.l.; 2011.
423
3. Byrd JH, Peace MR. Entomotoxicology: Drugs, Toxins, and Insects. In: Kobilinsky L, editor.
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Forensic Chemistry Handbook2011. p. 483-99.
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4. Carvahlo LML. Toxicology and forensic entomology In: Amendt J, Campobasso CP, Goff
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ML, Grassberger M, editors. Current Concepts in Forensic Entomology: Springer Science; 2010. p.
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163–78.
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5. Introna F, Campobasso C, Goff M. Entomotoxicology. Forensic Sci Int. 2001;120:42-7.
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6. Magni PA, Pacini T, Pazzi M, Vincenti M, Dadour IR. Development of a GC-MS method for
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methamphetamine detection in Calliphora vomitoria L. (Diptera: Calliphoridae). Forensic Sci Int.
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2014;241:96-101.
432
7. Morris H, Wallach J. From PCP to MXE: A comprehensive review of the non-medical use of
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dissociative drugs. Drug testing and analysis. 2014;6 (7-8):614-2.
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8. Corssen G, Domino EF. Dissociative anesthesia: further pharmacologic studies and first
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9. Kelly K. A little book of Ketamine: Ronin; 1999.
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join action on new synthetic drugs. Luxembourg: Office for Official Publications of the European
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Communities; 2002.
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11. AA.VV. Federal register1999.
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12. AA.VV. Ketamine. Factsheet. Australian Drug Foundation; 2014.
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13. Lalonde BR, Wallage HR. Postmortem Blood ketamine distribution in two fatalities J Anal
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Toxicol. 2004;28(1):71-4.
14. Albright JA, Stevens SA, Beussman DJ. Detecting ketamine in beverage residues:
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Application in date rape detection. Drug testing and analysis. 2012;4(5):337-41.
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15. Salvagni FA, de Siqueira A, Fukushima AR, Landi MF, Ponge-Ferreira H, Maiorka PC.
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2016;267:e1-e5.
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forensic relevance. J Forensic Sci. 2014;59(4):991-6.
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18. Zou Y, Huang M, Huang R, Wu X, You Z, Lin J, et al. Effect of ketamine on the
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development of Lucilia sericata (Meigen) (Diptera: Calliphoridae) and preliminary pathological
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observation of larvae. Forensic Sci Int. 2013;226(1-3):273-81.
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19. Derelanko MJ, Hollinger MA. Handbook of toxicology. Boca Raton, Florida: CRC Press;
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21. Smith KGV. A Manual of Forensic Entomology. London: Trustees of the British Museum,
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Natural History and Cornell University Press; 1986.
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22. Byrd JH, Castner JL. Insects of forensic importance. In: Byrd JH, J.L. C, editors. Forensic
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entomology—the utility of arthropods in legal investigation. Boca Raton, FL: CRC Press; 2010. p.
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39-126.
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23. Gennard D. Forensic Entomology. An Introduction. 2 ed: Wiley-Blackwell; 2012.
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24. Magni PA, Massimelli M, Messina R, Mazzucco P, Di Luise E. Entomologia Forense. Gli
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insetti nelle indagini giudiziarie e medico-legali: Ed. Minerva Medica; 2008.
25. Magni PA, Pazzi M, Vincenti M, Alladio E, Brandimarte M, Dadour IR. Development and
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validation of a GC–MS method for nicotine detection in Calliphora vomitoria (L.) (Diptera:
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Calliphoridae). Forensic Sci Int. 2016;261:53-60.
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26. Donovan S, Hall M, Turner B, Moncrieff C. Larval growth rates of the blowfly, Calliphora
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vicina, over a range of temperatures. Med Vet Entomol. 2006;20:106-14.
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27. Anderson G. Minimum and maximum development rates of some forensically important
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Calliphoridae (Diptera). J Forensic Sci. 2000;45:824-32.
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28. Wolff K, Winstock AR. Ketamine: from medicine to misuse CNS Drugs. 2006;20(3):199-218
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29. Amendt J, Campobasso CP, Gaudry E, Reiter C, LeBlanc HN, Hall M. Best practice in
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forensic entomology. Standards and guideline. Int J Legal Med. 2007;121:104-9.
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30. Day D, Wallman J. Width as an alternative measurement to length for post-mortem interval
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estimations using Calliphora augur (Diptera: Calliphoridae) larvae. Forensic Sci Int.
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31. Bourel B, Tournel G, Hedouin V, Deveaux M, Goff M, Gosset D. Morphine extraction in
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necrophagous insects remains for determining ante-mortem opiate intoxification. Forensic Sci Int.
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32. Raposo F. Evaluation of analytical calibration based on least-squares linear regression for
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instrumental techniques: a tutorial review. TrAC Trends in Analytical Chemistry. 2016;77:167-85.
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33. AA.VV. International Conference on Harmonisation of Technical Requirements for
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Registration of Pharmaceuticals for Human Use. Validation of analytical procedures: text and
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34. AA.VV. ISO/IEC 17025:2005. General requirements for the
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competence of testing and calibration laboratories. 2 ed: International Organization for
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35. Olivieri AC. Practical guidelines for reporting results in single- and multi-component
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analytical calibration: a tutorial. Analytica Chimica Acta. 2015;868:10-22.
497
36. Mandel J. The statistical analysis of experimental data. New York: Dover Pubblication;
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37. Hubaux A, Vos G. Decision and detection limits for linear xalibration curves. Analical
500
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38. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix
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effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem. 2003;75:3019-30.
503
39. Liu Y, Lin D, Wu B, Zhou W. Ketamine abuse potential and use disorder. Brain Res Bull.
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40. Administration SAMHS. The NSDUH Report Use of Specific Hallucinogens: 2006. Office of
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41. Morgan CJ, Curran, H.V., Independent Scientific Committee on Drugs. Ketamine use: a
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509
42. Goff ML, Lord WD. Entomotoxicology – Insects as toxicological indicators and the impact of
510
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Utility of Arthropods in Legal Investigations. 2 ed: CRC Press; 2010. p. 427–36.
512
43. Nortueva P, Nortueva SL. The fate of mercury in sarcosaprophagous flies and in insects
513
eating them. Ambio. 1982;11:34–7.
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44. Amendt J, Campobasso CP, Gaudry E, Reiter C, LeBlanc HN, Hall MJ. Best practice in
515
forensic entomology - Standards and guidelines. Int J Legal Med. 2007;121(2):90-104.
516
45. Liu X, Shi Y, Wang H, Zhang R. Determination of malathion levels and its effect on the
517
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46. Sukontason K, Piangjai S, Siriwattanarungsee S, Sukontason K. Morphology and
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47. Adams ZJO, Hall MJR. Methods used for the killing and preservation of blowfly larvae, and
523
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524
48. George K, Archer M, Green L, Conlan X, Toop T. Effect of morphine on the growth rate of
525
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Table 1 529
Triple quadruple monitored transitions and applied collision energy.
530 531
Substance Precursor Ion Fragment Ion Collision Energy (V) Use of transition
Ketamine 238 125 20 Quantitation 238 179 13 Identification 238 207 10 Identification 238 220 10 Identification Ketamine-d4 242 129 7.5 Identification 242 211 23.5 Identification 242 224 10 Quantitation 532 533
Table 2 534
Parameters calculated for ketamine using HPLC-MS/MS.
535 536 537 Parameter Value HPLC-MS/MS Coefficient of linearity, R2 0.99677
Limit of detection (ng/mg), LOD 0.015
Limit of quantitation (ng/mg), LOQ 0.031
Extraction recovery (%) at 0.75 ng/mg concentration 99.4
Extraction recovery (%) at 2 ng/mg concentration 100
CV % at 0.75 ng/mg concentration 14
CV % at 2 ng/mg concentration 16
538 539 540
Table 3 541
Ketamine quantitation (ng/mg ± S.E.) in C. vomitoria (L2=second instar, L3=third instar,
PF=post-542
feeding instar, P=pupa instar, EP=empty puparium, A=adult instar) detected through HPLC-MS/MS
543
analysis. Quantitation was calculated using 3 replicates. Ketamine LODHPLC-MS/MS=0.015 ng/mg and 544
LOQHPLC-MS/MS=0.031 ng/mg. The groups indicated in brackets (i.e. C, T1, T2) were significantly 545
different (P<0.05) from the group indicated in the corresponding column.
546 547
Treatment Control (C) T1 T2
Amount of ketamine
spiked with liver 0 ng/mg 300 ng/mg 600 ng/mg
S a m pl ing d ay (Li fe ins tar ) Day 3
(L2) <LOD <LOD <LOD
Day 4 (L3) <LOD (T1, T2) 14.9 ± 0.03 (C, T2) 180.0 ± 0.28 (C, T1) Day 5 (L3) <LOD (T1, T2) 7.40 ± 0.03 (C, T2) 16.9 ± 0.24 (C, T1) Day 7 (PF) <LOD (T1, T2) 0.15 ± 0.02 (C, T2) 0.97 ± 0.19 (C, T1) Day 9 (PF) <LOD (T1, T2) 0.05 ± 0.02 (C, T2) 0.35 ± 0.23 (C, T1) Day 11 (P) <LOD (T1, T2) 0.20 ± 0.28 (C, T2) 0.81 ± 0.11 (C, T1) EP <LOD (T1, T2) 1.00 ± 0.08 (C, T2) 2.06 ± 0.21 (C, T1)
A <LOD < LOD < LOD
548 549 550
Table 4 551
Time (days ± S.E.) from oviposition to pupation and to eclosion of C. vomitoria larvae, which were
552
exposed to either liver containing different amount of ketamine, or to the control liver. The table
553
shows also the number of larvae dead prior to pupation, the number of not emerged adults, and
554
the number of survivals. The groups indicated in brackets (i.e. C, T1, T2) were significantly
555
different (P<0.05) from the group indicated in the corresponding column.
556 557
Treatment Control (C) T1 T2
Amount of ketamine spiked with liver 0 ng/mg 300 ng/mg 600 ng/mg
Larvae third instar N= 100 100 100
Time (days) from oviposition to pupation 9.89 ± 0.13
(T2) 10.30 ± 0.10
10.46 ± 0.09 (C)
Larvae dead prior to pupation 2 10 15
Pupae 98 (T1, T2) 90 (C) 85 (C)
Pupae % 98% 90% 85%
Pupae N= 98 90 85
Time (days) from oviposition to eclosion 18.40 ± 0.10 (T1, T2)
19.27 ± 0.18 (C)
20.00 ± 0.10 (C)
Not emerged adults 17 57 77
Survival 81 (T2, T3) 33 (C, T2) 8 (C, T1) Survival % 83% 37% 9% 558 559 560
Table 5 561
C. vomitoria larvae and pupae mean lengths (mm ± S.E.) related to instar of life (L2=second instar,
562
L3=third instar, PF=post-feeding instar, P=pupa instar). The groups indicated in brackets (i.e. C, T1,
563
T2) were significantly different (P<0.05) from the group indicated in the corresponding column. For
564
each time of exposure and each treatment N=30.
565 566
C. vomitoria means length (mm ± S.E.)
Treatment Control (C) T1 T2
Amount of ketamine spiked with liver 0 ng/mg 300 ng/mg 600 ng/mg
S a m pl ing day (Li fe ins tar ) Day 3 (L2) 6.56 ± 0.25 6.79 ± 0.21 7.19 ± 0.16 Day 5 (L3) 16.87 ± 0.15 16.54 ± 0.25 17.20 ± 0.29 Day 6 (L3) 16.79 ± 0.26 (T2) 17.25 ± 0.27 (T2) 18.50 ± 0.23 (C, T2) Day 7 (PF) 11.91 ± 0.24 12.59 ± 0.31 12.45 ± 0.28 Day 8 (PF) 11.56 ± 0.21 11.57 ± 0.13 11.69 ± 0.28 Day 11 (P) 9.27 ± 0.11 (T2) 9.49 ± 0.09 9.82 ± 0.12 (C) 567 568 569
Table 6 570
C. vomitoria larvae and pupae mean widths (mm ± S.E.) related to instar of life (L2=second instar,
571
L3=third instar, PF=post-feeding instar, P=pupa instar). The groups indicated in brackets (i.e. C, T1,
572
T2) were significantly different (P<0.05) from the group indicated in the corresponding column. For
573
each time of exposure and each treatment N=30.
574 575
C. vomitoria mean width (mm ± S.E.)
Treatment Control (C) T1 T2
Amount of ketamine spiked with liver 0 ng/mg 300 ng/mg 600 ng/mg
S a m pl ing d ay (Li fe ins tar ) Day 3 (L2) 0.89 ± 0.04 (T2) 1.03 ± 0.02 1.05 ± 0.03 (C) Day 5 (L3) 2.31 ± 0.50 2.30 ± 0.06 2.37 ± 0.07 Day 6 (L3) 2.33 ± 0.07 (T1, T2) 2.70 ± 0.06 (C) 2.86 ± 0.05 (C) Day 7 (PF) 2.51 ± 0.07 (T2) 2.39 ± 0.06 (T2) 2.92 ± 0.04 (C, T1) Day 8 (PF) 2.26 ± 0.04 (T2) 2.39 ± 0.05 (T2) 2.70 ± 0.05 (C, T1) Day 11 (P) 2.88 ± 0.05 (T2) 3.08 ± 0.04 3.10 ± 0.06 (C) 576 577 578
